Recently, the process of analyzing, purifying, and detecting proteins and other macromolecules has become a staple technology in the fields of biotechnology, drug discovery, therapeutics, clinical analysis, chemical analysis, and the like. Accordingly, materials and methods capable of effectively identifying a target protein or the like are highly awaited.
To date, to meet such a need, a method has been proposed for producing a molecularly imprinted polymer which has an imprint of a protein, in which the polymer is produced by radical polymerization of a monomer and a crosslinker in the presence of the protein (Non-patent document 1). This method, however, does not disclose any particular technique to synthesize the polymer with optimal molecular identifying capacity for a particular protein, and thus, trial and error experimentation is necessary for the implementation of this method.
Most molecularly imprinted polymers are prepared in an organic solvent, such as acetonitrile or chloroform, and show selectivity for small molecules, such as sugars, amino acids, and steroids (Non-patent documents 2 to 4). More specifically, these methods use a small molecule as template to synthesize a molecularly imprinted polymer in an organic solvent. Accordingly, these methods cannot be applied to the synthesis of a molecularly imprinted polymer to be used for capturing a water-soluble macromolecule, such as a protein, wherein the synthesis has to be conducted in an aqueous solution.
Accordingly, many researchers are trying to synthesize a molecularly imprinted polymer which can selectively identify a macromolecule in an aqueous solution.
For example, researchers have attempted to entrap a protein within polysiloxane or polyacrylamide in a buffer solution to thereby form the molecularly imprinted polymer (Non-patent Documents 5 to 8). This attempt, however, suffered from the problems of insufficiently reproduced protein shape, insufficient strength and softness of the polymer, and low selectivity. Another attempt was made with the use of polyaminophenylboronic acid to embed peroxidase in the disposable microtiter plate surface coating (Non-patent Document 9).
In a more recent attempt, a molecularly imprinted polymer was synthesized by a method called “surface-imprinting procedure”, to selectively capture the protein (Non-patent Documents 10 and 11). This method, involving the use of a metal (Cu2+) chelating monomer, however, could be applied only to proteins having the special structure with histidine residue being exposed on the surface.
Another attempt involved the use of a cyclodextrin-based functional monomer for the formation of the polymer imprinted with a polypeptide molecule in an aqueous solution (Non-patent Documents 12 and 13). This method, however, is applicable only to low molecular weight compounds such as dipeptides (Phe-Phe).
Meanwhile, the inventors of the present invention have discovered an approach called “epitope approach”. In this approach, a polymer imprinted with the shape of a lower molecular weight protein, which is different from the target protein but whose main structure comprises a part of the target protein, is synthesized in an organic solvent, and the target protein having the higher molecular weight is selectively entrapped by using this polymer (Patent Document 1).
[Patent Document 1] Unexamined Published Japanese Patent Application No. (JP-A) 2001-55399
[Non-patent Document 1] Angew. Chem., Int. Ed. Engl. 1995, vol. 34, pp. 1812-1832
[Non-patent Document 2] Clin. Chem., 42(1996) 1506
[Non-patent Document 3] Nature, 361(1993), 645
[Non-patent Document 4] Proc. Nat. Acad. Sci., USA 92(1995) 4788
[Non-patent Document 5] Biochim. Biophys. Acta, 1250(1995) 126
[Non-patent Document 6] Biochim. Biophys. Res. Commun., 227(1996) 419
[Non-patent Document 7] Chromatographia, 44(1997) 227
[Non-patent Document 8] Chem. Lett., (1998) 731
[Non-patent Document 9] Anal. Chem., 73(2001) 5281
[Non-patent Document 10] J. Mol. Recogn., 8(1995) 35
[Non-patent Document 11] J. Amer. Chem. Soc., 123(2001) 2072
[Non-patent Document 12] J. Mol. Recogn., 11(1998) 94
[Non-patent Document 13] Anal. Chim. Acta, 435(2001) 25